A Tracer of Life: In Lindau, all three 2008 Chemistry Nobel Laureates will report on their research with fluorescent pro

Today, nearly every area of the biomedical sciences - from infectious diseases research to neuroscience and oncology - benefits from the discovery of the green fluorescent protein (GFP) and its analogues, a class of proteins whose scientific history began 49 years ago and continues to this day. The 600 young researchers from 66 countries who will be participating in next week's Lindau Nobel Laureate Meeting (June 28th - July 3rd) can experience this history first-hand. Along with 20 other Nobel Laureates, all three of last year's chemistry prizewinners will be coming to Lake Constance to report on their work: Osamu Shimomura, Martin Chalfie and Roger Tsien.
Getting a jellyfish from the Pacific Ocean to divulge its secret, and thereby obtaining insights into hitherto invisible life processes for the entire field of biology - this is a superb example of the value of basic research and its initially unforeseeable implications. Today, nearly every area of the biomedical sciences - from infectious diseases research to neuroscience and oncology - benefits from the discovery of the green fluorescent protein (GFP) and its analogues, a class of proteins whose scientific history began 49 years ago and continues to this day. Passion and perseverance, chance and circumstance, patience and genius are all part of this story whose three protagonists - Osamu Shimomura, Martin Chalfie and Roger Tsien - were awarded the Nobel Prize in Chemistry last year. The 580 young researchers from 67 countries who will be participating in next week's Lindau Nobel Laureate Meeting can experience this history first-hand. Along with 20 other Nobel Laureates, all three of last year's prizewinners will be coming to Lake Constance to report on their work.

Some sea creatures glow. Particularly impressive are the glowing green spots on the bell margin of the jellyfish Aequorea victoria, a species that is found off the west coast of North America. In summer 1960, Princeton University biology professor Frank Johnson instructed his assistant, Osamu Shimomura, to investigate this phenomenon. During the following 19 years, Shimomura and his staff fished some 850,000 jellyfish from the Pacific. Initially, the researchers drew a filtrate from the bell margin of the jellyfish that, to their surprise, emitted a blue light. In an effort spanning several months, Shimomura managed to extract a few drops of a blue fluid, and to isolate the intact luminescent protein that this fluid contained. He called it 'aequorin'. The protein produced flashes of light in the presence of calcium ions, which are in abundant supply in ocean water. Why, though, was the light that the intact jellyfish emitted green-coloured? Because it contains a second protein that absorbs and transforms the blue light. This second protein is GFP: a light-absorber/emitter in the form of a molecule known as a chromophore. It causes the protein to fluoresce in green by reflecting incoming ultraviolet and blue light in green-coloured wavelengths, as Shimomura described in 1979 after isolating one-tenth of a milligram of this chromophore from 100 milligrams of GFP.

At the time, it was still too early to specify these findings further, or to put them to practical use in the field. Only with the rapid progress made in gene technology could Douglas Prasher, beginning in the mid-1980s, uncover the genetic blueprints, first of aequorin and, later, of GFP and thus identify their structures. GFP consists of 238 amino acids folded in such a way that the molecule resembles a beverage can, with three amino acids running through the centre and responsible for providing the characteristic colour of the chromophore.

In 1988, Martin Chalfie began to take an interest in GFP as well. He first learned of GFP in a seminar about luminescent organisms held at Columbia University in New York; to him, the accounts were electrifying. His specialty was the nematode C. elegans, which his former supervisor Sidney Brenner (Nobel Prize in Medicine, 2002) had introduced to molecular biology as a model organism. While C. elegans consists of a mere 959 cells, it nevertheless has a brain, it ages and reproduces. One third of its genes - the blueprints for its proteins - are related to genes found in humans. In addition, the species is transparent. Chalfie knew that its organs would be easier to study under a microscope if they were fluorescent. He came up with the following idea: if I could couple the gene for GFP to a gene specific to a particular organ in the nematode, then this combined blueprint would result in a protein that would cause the specific organ to luminesce in green. With the help of the head of his seminar, Chalfie contacted Douglas Prasher, who was working on decoding the GFP gene at the time, and who was then able to send a copy of the gene to Chalfie in 1992. Chalfie now coupled it with the gene for a protein that is only active in the nematode's six touch receptor neurons. In 1994, the results of this experiment were emblazoned across the title page of Science magazine: a nematode that had bright green bioluminescent nerve receptors.

With this basic experiment, Chalfie had demonstrated that, aside from ultraviolet or blue light, no other energy source or enzymes are required to activate GFP. Moreover, GFP had not caused any damage to the nematode. It could also be applied without difficulty to the bacterium E. coli, to the fruit fly, or to baker's yeast, the other key model organisms for molecular biologists. Apparently, GFP was suitable as a universal genetic marker. The resounding impact of this discovery on experimental biomedicine is reflected in the more than 20,000 papers published on GFP since Chalfie first reported his findings.

It is due to the work of Roger Tsien that, from today's viewpoint, GFP is only the point of departure for an entire palette of luminescent colours that can be applied in biology and that are also suitable for use in mammalian cells. Tsien analyzed the precise mechanism of colour transformation in the chromophore of GFP and found that this only takes place in the presence of oxygen. Using pinpointed gene-technological procedures to replace various amino acids in the chromophore, Tsien was able to construct variants that absorb and emit light from different portions of the colour spectrum. His research also drew on suggestions of two Russian researchers who had discovered proteins similar to GFP in a luminescent coral. As a result, cells and individual proteins can now be marked in all colours of the rainbow - some 100 different hues have been described.

Targeted coupling of proteins on luminescent markers similar to GFP makes it possible to view 'live' scenes under the microscope - mitosis in individual stages of cell division, the interactions between signal molecule and receptor, or the formation of new viruses in infected cells.

Proteins are the fundamental molecules of life: they maintain its structures while controlling its dynamics. Their structures have already been largely illuminated in atomic detail by the methods of x-ray technology or nuclear magnetic resonance spectroscopy (NMR). Prize-winning pioneers in x-ray crystallography, such as Hartmut Michel and Robert Huber (Nobel Prize, 1988), who used this method to elucidate the first three-dimensional structure of a membrane protein; and in magnetic resonance spectroscopy, such as Richard Ernst (Nobel Prize, 1991) and Kurt Wüthrich (Nobel Prize, 2002), will also participate in this year's meeting in Lindau. Yet monitoring proteins' dynamics within living cells using the methods of structural elucidation is only possible by sequenced still images. GFP-based methods, on the other hand, cannot elucidate structures - but they allow analyses of the dynamics of single molecules within a single cell as well as of entire tissues.

Roger Tsien continues to work intensively on developing new areas of application for these methods. Researchers on his team recently reported on a new molecule that fluoresces infrared light - a development that could overcome one of the current limitations of luminescent markers. This is because their visible light is absorbed by some organs or scattered by bone tissue. Infrared fluorescence, on the other hand, has enabled examination of the liver of a live mouse which in turn permits gentler treatment of laboratory animals. Tsien's long-term objective is to generate luminescent molecules that can be activated without genetic transfer. These could then be used in human patients, where they would serve as clinical biomarkers for the diagnosis and monitoring of diseases.
Weitere Informationen:
http://www.lindau-nobel.de/2009_Meeting_Chemistry.AxCMS?ActiveID=1338 - Information about the 2009 Nobel Laureate Meeting (Abstracts, Participants, Programme)
http://www.lindau-nobel.de - Live-Webstreams of Lectures and Panels
http://www.scienceblogs.de/lindaunobel - Official Conference Blog
http://www.twitter.com/lindaunobel - News live from the meeting

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